Reduction in Xenogeneic Epitopes on Porcine Endothelial Cells by Periodate Oxidation.

BACKGROUND

Patterns of humoral immune responses represent a major hurdle in terms of pig-to-human xenotransplantation approaches. The best-known xenogeneic glycan antigens present in pigs are the αGal (Galili antigen) and the non-human sialic acid Neu5Gc. As there are further differences between porcine and human cellular surface glycosylation, a much broader range of glycan epitopes with xeno-reactive relevance can be anticipated. Therefore, we set out to chemically modify porcine cellular surface glycans in a global approach by applying sodium periodate (NaIO) oxidation.

METHODS

Porcine endothelial cells were exposed to oxidation with 1 to 5 mM NaIO for different time periods at 37 °C or 4 °C and under static or dynamic conditions. The impact on cellular survival was determined by applying live/dead assays. Oxidation of αGal-epitopes was assessed by fluorescence microscopy-based quantification of isolectin-B4 (IL-B4) staining. Overall immunogenicity of porcine cells was determined by human serum antibody binding.

RESULTS

Treatment of porcine endothelial cells and tissues with NaIO led to reduced binding of the αGal-specific IL-B4 and/or human serum antibodies. NaIO was revealed to be cytotoxic when performed at elevated temperatures and for a prolonged time. However, by applying 2 mM NaIO for 60 min at 4 °C, a high extent of cellular viability and a relevant reduction in detectable αGal epitope were observed. No differences were detected irrespectively on whether the cells were oxidized under static or flow conditions.

CONCLUSIONS

Glycan epitopes on living cells can be oxidized with NaIO while maintaining their viability. Accordingly, this strategy holds promise to prevent immune reactions mediated by preformed anti-glycan antibodies.